Macromolecularized adenine derivatives

ABSTRACT

A method is disclosed for preparing functionalized adenine derivatives, said method comprising the step of reacting a compound which contains an adenine nucleus which has been halogen substituted in the 8-position, with a compound having the general formula N +-  S--(CH 2 ) n  --CO0 -  M +  wherein M +  is the ion of an alkali metal and n is an integer, the reaction being carried out in a polar aprotic solvent. Procedures for preparing macromolecularized adenine compounds are also indicated by reacting a functionalized adenine derivative with a polymer which has at least one primary or secondary aminic group in its structure.

This is a division of application Ser. No. 705,012, filed July 14, 1976,now U.S. Pat. No. 4,091,203.

This invention relates to a method for the preparation of functionalizedadenine derivatives and to the products obtained thereby. Moredetailedly, the present invention relates to a method for thepreparation, starting from compounds which contain an adenine nucleushaving a halogen atom in the 8-position, of functionalized adeninederivatives carrying in said position an omega-carboxylic side chain.The starting materials for said method can be obtained, withconventional methods, by halogenating compounds which contain theadenine nucleus, such for example nicotinoylamine adenine dinucleotide(NAD⁺), nicotinoylamide-adenine dinucleotide phosphate (NADP⁺),adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosinemonophosphate (AMP), adenosine.

The majority of these compounds have an outstanding importance inbiochemistry and their functionalization widens their field ofapplication.

For example, in the case of NAD⁺, but these considerations hold goodalso for the other members, its functionalized derivatives can be used,after having been attached by a covalent bond to macromolecules whichare either water-soluble or water-insoluble, as non-diffusiblecoenzymes, or in affinity chromatography. Thus, in the case ofattachment to water-soluble macromolecules, they can be used as nondiffusible, water-soluble macromolecularized coenzymes. These permit thewidening of the field of application of the known enzymic systems inwhich the enzyme is physically embedded in insoluble porous structures,such as fibers, polyacrylamide gel, microcapsules, etc. which areimpervious to macromolecules. As a matter of fact, by physicallyembedding, together with the enzyme or a polyenzymic system also thewater-soluble macromolecularized coenzyme, both the enzyme and thecoenzyme remain in close contact and the scattering of the lattertowards the outside of the occluding structure is prevented, while withthe natural coenzyme this cannot be done on account of the low molecularweight of the latter. In the case of attachment of water-insolublemacromolecules they can be used for affinity chromatography or forenzymic reactions in a heterogeneous phase, it being possible to recoverthe coenzyme.

According to the present invention, the above mentioned derivativeswhich have been functionalized in the adenine nucleus are obtained byreacting the corresponding starting compound which has beenhalogen-substituted at the C₈ of the same nucleus, with the di-salt ofan omega-mercaptocarboxylic acid having the general formula:

    M.sup.+- S--(CH.sub.2).sub.n --COO.sup.- M.sup.+

wherein M⁺ is the ion of an alkali metal and n is an integer.

The reaction is conducted in aprotic polar solvents (such ashexamethyl-phosphotriamide, dimethyl sulphoxide and dimethylformamide)at a temperature ranging from +20° C. to +60° C., preferably at roomtemperature, and under anhydrous conditions.

The reaction causes a substitution of the halogen in the 8-position ofthe adenine nucleus in the manner shown by the formula: ##STR1## whereinn and M⁺ have the meanings as indicated above, X is a halogen and R isthe non-adenine residue of the compound.

The salt of the as-obtained carboxyl derivative is then converted duringprogress of the processing, into its corresponding free acid.

The method is absolutely general, but in the following portion of thedisclosure, reference will be had to the reaction of the sodium bi-saltof the 3-mercaptopropionic acid with the derivatives of NAD⁺ and NADP⁺which have been brominated at the 8-position carbon of the adeninenucleus of adenosine, with the aim of illustrating the methods which arerequired for carrying out said process.

It will be anyhow apparent, on reading the following, that anyoneskilled in the art will be able to obtain functionalized adeninederivatives of the kind referred to above, starting from any 8-halogenadenine substrate by merely adapting the working conditions to thenature of the starting compound, without departing from the scope of thepresent invention.

This invention is also concerned with the preparation ofmacromolecularized adenine derivatives and the products obtainedthereby, these latter containing one or more units having the generalformula: ##STR2## wherein n is an integer, and the radical ##STR3## canbe obtained from any compound having adenine nucleus, such as, forexample, nicotinoylamide-adenine dinucleotide, nicotinoylamide-adeninedinucleotide phosphate, adenosine monophosphate, cyclic adenosinemonophosphate, adenosine biphosphate, adenosine triphosphate, adenosine,adenine and wherein the nitrogen atom bound to the CO group is a part ofa compound having a high molecular weight and which is eitherwater-soluble or water-insoluble and contains one or more primary orsecondary aminic groups (for example: polylysine,omega-aminoalkylpolyacrylamides, polysaccharide esters ofomega-aminoalkylcarbamic acids, polyvinylamine, omega-aminoalkyl esters,or omega-aminoalkyl amides of the polyglutamic acid, aminoalkylsilanizedglass microspheres, polyethyleneimine and others). Such functionalizedcompounds can react with at least one polymer having at least a primaryor secondary aminic group, to give the macromolecularized adeninederivatives mentioned above, in the presence of a water-solublecarbodiimide (for exampleN-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride) or aninsoluble one (for example: N,N'-dicyclohexylcarbodiimide) as acondensing agent.

The condensation reaction between the carboxyl group of thefunctionalized adenine derivative and the aminic group of themacromolecule to give the amidic bond, is carried out in an aqueousenvironment or in a mixture of water and a water-soluble organic solvent(for example: pyridine, tetrahydrofuran, dioxan and others) at atemperature comprised between +5° C. and +50° C.; and preferably at roomtemperature.

The macromolecularized adenine derivatives which are the subject-matterof the present invention, have a number of applications.

For example, in the case of the macromolecularized derivatives of thenicotinoylamide-adenine dinucleotide (NAD), but the same is true of theother macromolecularized derivatives, they find application in affinitychromatography or as active, non-diffusible coenzymes.

Thus, in the case of attachment to water-soluble macromolecules, theycan be used as non-diffusible, water-soluble macromolecularizedcoenzymes. These permit the widening of the field of application of theknown enzymic systems in which the enzyme is embedded physically ininsoluble structures, such as fibers, polyacrylamide gels, microcapsulesand others, which are impervious to the macromolecules. As a matter offact, by physically embedding together with the enzyme, or thepolyenzymic system, also the water-soluble macromolecularized coenzyme,both the enzyme and the coenzyme remain in close contact and thescattering of the coenzyme towards the outside of the occludingstructure is prevented, whereas, with the natural coenzyme, this cannotbe done due to its low molecular weight.

In the case of attachment to water-insoluble macromolecules, they can beused for affinity chromatography or for enzymic reactions in aheterogeneous phase, it being possible to recover the coenzyme.

EXAMPLE No. 1

Preparation of the 8-(2-carboxyethylthio)adenosine.

To a solution of 380 milligrams (1.1 millimol) of 8-bromoadenosine in 5mls of anhydrous phosphotriamide there are added with stirring at roomtemperature and in an anhydrous atmosphere (nitrogen), 633 milligrams(4.2 millimols) of the sodium disalt of 3-mercaptopropionic acid(obtained by treating at room temperature the mercaptoacid with astoichiometric amount of sodium hydride in anhydrous tetrahydrofuran andsubsequent withdrawal of the solvent under vacuum).

After 16 hours of stirring at room temperature, the mixture is filteredand the filtrate is supplemented with 15 mls of water and extractedseveral times with chloroform until hexamethylphosphotriamide has beendischarged.

The aqueous solution adjusted to a pH 8.5 with diluted HCl, ischromatographed on DOWEX-1 (HCOO⁻) eluting with a gradient of formicacid in water. The fractions with λ_(max) 282 mm are combined andfreeze-dried to give 327 milligrams of 8-(2-carboxyethylthio)-adenosine.

The product proves to be pure at thin layer analysis (silica gel with afluorescence indicator; eluent; isopropanol--water--32% ammonia in thevolume ratios of 7:2:1; visualization of the spot by a UV lamp at 254nm; Rf=0.56) and also at the high-voltage electrophoresis (Whatman paper3 MM 11×57 cm; electrolyte 0.02 M ammonium acetate pH 5.0, potential5000 V during 40 mins., visualization of the spot with a UV lamp at 254nm; the mobility of the product towards the anode is in agreement withthe presence of the carboxyl group whereas adenosine migrates towardsthe cathode).

The UV spectrum in 0.1 M NaOH exhibits peak absorbance at 282 nm whereasthat of 8-bromoadenosine is at 263 nm.

The ¹ H NMR in NaO² H shows, in addition to the signals relative toadenosine with the exclusion of that of the proton at the 8-position,those relative to the protons of the side chain: 2.88 (2H, t; CH₂ COO)and 3.86 (2H, t; CH₂ S).

Also the mass spectrum confirms the structure attributed to the product(m/e 239, 221, 192, 167).

EXAMPLE No. 2

Preparation of the nicotinoylamide-8-(2-carboxyethylthio)adeninedinucleotide.

To 118 milligrams (160 micromols) of nicotinoylamide-8-bromoadeninedinucleotide dissolved in 2 mls of anhydrous dimethylsulphoxide thereare added with stirring at room temperature and under an anhydrousatmosphere (nitrogen), 98.4 milligrams (656 micromols) of the sodiumdisalt of the 3-mercaptopropionic acid (prepared as described in Example1).

After 16 hours of stirring at room temperature, the mixture is filteredand ten volumes of acetone are added to the filtrate.

The obtained precipitate, separated by centrifugation and washed withacetone, is dried in a vacuo and dissolved in 15 mls of 0.1 M HCl. Thesolution, as adjusted to a pH of 7.5 with diluted soda, ischromatographed on DOWEX-1 (HCCO⁻) by eluting with a gradient of formicacid in water.

The chromatographic fractions with λ_(max) 276.5 are combined andfreeze-dried to give 90 milligrams of nicotinoylamide8-(2-carboxyethylthio)adenine dinucleotide. The product proves to bepure at the thin layer analysis silica gel with fluorescence indicator;eluent; isobutyric acid--water--32% ammonia in the volume ratios of66:33:1.7; visualization of the spot with an UV lamp at 254 nm; Rf0.31), and at high-voltage electrophoresis (3 MM Whatman paper 11 by 57cm; electrolyte: 0.02 M ammonium acetate, pH 5.0, potential 5,000 Vduring 30 minutes; visualization of the spot by UV lamp at 254 nm;mobility towards the anode greater than that of NAD⁺, consistently withthe presence of the carboxyl group).

The Ultraviolet spectrum in a solution of pyrophosphate buffer, pH 8.7,shows a peak at 276.5 nm which, by enzymic reduction with alcoholdehydrogenase from yeast, passes to 282 nm with the appearance of a newpeak at 340 nm which is characteristic of the reduced nicotinoylamidenucleus.

The ¹ H NMR in ² H₂ O shows, in addition to the signals relative to NAD⁺with the exclusion of that of the proton attached to the carbon in the8-position of the adenine nucleus, those relative to the protons of theside chain: δ2.88 (2H, t; CH₂ COO) and 3.86 (2H, t; CH₂ S). Also themass spectrum is in agreement with the structure attributed to theproduct (m/e 221, 192, 167).

EXAMPLE No. 3

Preparation of the nicotinoylamide-8-(2-carboxyethylthio)adeninedinucleotide phosphate.

To 50 milligrams (57.6 micromols) of nicotinoylamide-8-bromoadeninedinucleotide phosphate dissolved in 1 ml of anhydrousdimethylsulphoxide, there are added with stirring at room temperatureand in an anhydrous atmosphere (nitrogen), 36 milligrams (240 micromols)of the sodium disalt of the 3-mercaptopropionic acid (prepared asdescribed in Example 1).

After 16 hours of stirring at room temperature, the mixture has beenfiltered and the filtrate is supplemented with ten volumes of acetone.The as-obtained precipitate, separated by centrifugation and washed withacetone, is dried in a vacuo and dissolved in 10 mls of 0.1 M HCl.

The solution, adjusted to pH 7.5 with diluted soda, is chromatographedon DOWEX-1 (Cl⁻) by eluting with a gradient of CaCl₂ in water which hasa pH of 3 by addition of HCl. The chromatographic fractions containingthe expected product are combined, concentrated to a small volume, anddesalified by gel-filtration on Sephadex G-10 by eluting the productwith water.

The characterization of thenicotinoylamide-8-(2-carboxyethylthio)adenine dinucleotide phosphate iscarried out similarly to what has been described in Example 2 for thecorresponding derivative of the NAD⁺ (glucose-6-phosphate dehydrogenasehas been used for the enzymic reduction).

EXAMPLE No. 4

Preparation of thenicotinoylamide-8-(polyethyleneimine-3-carbonylethylthio)adeninedinucleotide. ##STR4##

To 0.76 mls of an aqueous solution of polyethyleneimine (33% conc.weight/volume) adjusted to a pH of 5 with conc.HCl, there are added 39milligrams of nicotinoylamide-8-(carboxyethylthio)adenine dinucleotide##STR5## dissolved in 0.5 ml of water and 40 milligrams ofN-ethyl-N'-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride dissolvedin 0.5 mls of water.

The reaction mixture, adjusted to a pH of 4.8 by addition of 2 M HCl isstirred at room temperature during 48 hours maintaining the pH at 4.8during the first three hours by addition of 0.1 M HCl. The reactionmixture diluted with water to 20 mls is then transferred to acentrifugation tube and precipitated with 20 mls of 1 M phosphate bufferat a pH of 6. Centrifugation for 10 minutes is effected at 39,000 g andthe solution is stripped of the polymeric precipitate by decantation.

In order further to purify the polymer, the latter is dissolved in 4 mlsof a 2 M solution in NaCl and 0.05 M in acetate buffer at a pH of 5.5and the solution thus obtained is supplemented by 16 mls of water andprecipitated again with 20 mls of 1 M phosphate buffer at pH 6 andcentrifuged during 10 minutes at 39,000 g by collecting the polymer bydecantation.

Such a purification procedure is repeated for at least four times.

The product redissolved in a 2 M solution in NaCl and 0.5 M in acetatebuffer at pH 5.5, is dialized against portions of 11 of 1.10⁻⁴ M HClduring four days, changing the solution every day.

By freeze drying the residue of the dialysis, there are obtained 223milligrams of the polymeric derivative of NAD, λ_(max) 276.5 nm.

The determination of the total NAD bound to the polymer is carried outby the measurement of the absorbance at 276.5 nm by assuming anextinction coefficient equal to that of the NAD derivative II (ε18800M⁻¹ cm⁻¹).

The determination of the coenzymically active NAD bound to the polymeris effected by quantitative enzymic reduction with alcohol dehydrogenasefrom yeast in a 0.1 M tris buffer at pH 9 in the presence of 0.2 Methanol and 0.5 M semicarbazide hydrochloride.

From the spectrophotometric measure at 340 nm of NADH derivative whichhas been formed, the result is that 115 micromols of enzymicallyreducible NAD are bound to each gram of the polymer, and correspond to90% of the total NAD bound to the polymer.

The thus obtained macromolecularized NAD is coenzymically active withseveral dehydrogenase. For example, with alcohol dehydrogenase fromyeast, the speed of enzymic reduction of the macromolecularized NAD is50% relative to that of the natural coenzyme. The determination iscarried out in the following incubation mixture (1.0 ml): tris. HCl, 83micromols, ethanol 166 micromols, semicarbazide. HCl, 42 micromols;coenzyme 0.1 micromol (expressed as bound and enzymically reducibleNAD); enzyme 0.2 micrograms; pH 9.0; incubation temperature 25° C. Thereduction speed is determined by the increase of the absorbance at 340nm.

EXAMPLE 5

Preparation of thenicotinoylamide-8-(polylysine-2-carbonylethylthio)adenine dinucleotide##STR6##

To 50 milligrams of polylysine hydrobromide having a mol.wt. of about50,000 and dissolved in 0.5 mls of water, there are added 40 milligramsof nicotinoylamide-8-(carboxyethylthio)adenine dinucleotide. ##STR7##dissolved in 0.5 ml of water, and 40 milligrams ofN-ethyl-N'-(3-dimethylaminopropyl)carbo diimide hydrochloride dissolvedin 0.5 ml of Water.

The reaction mixture, adjusted to a pH of 4.7 with 0.1 M NaOH is stirredat room temperature during 24 hours while maintaining the pH at 4.7during the first three hours by addition of 0.1 M HCl.

Under the same conditions there have been added 40 additional milligramsof carbodiimide in 0.5 ml of water and stirring is continued during 24additional hours.

The reaction mixture diluted with water to a volume of 15 mls is thentransferred to a centrifugation tube and precipitated with 10 mls of0.15 M phosphate buffer at a pH of 6.

Centrifugation is carried out during ten minutes at 19,000 g and thesolution of the polymeric precipitate is decanted.

To further purify the polymer, the latter is dissolved in 1 ml of a 2 Msolution in NaCl and 0.05 M in acetate buffer at pH 5.5 and the thusobtained solution is supplemented with 15 mls of water and precipitatedagain with 10 mls of 0.15 M, pH 6, pyrophosphate buffer and centrifugedduring 10 minutes at 39,000 g. collecting the polymer by decantation.

Such a purification procedure is repeated for at least four times. Theproduct, redissolved in a 2 M solution in NaCl and 0.05 M in pH 5.5acetate buffer, is dialized during 48 hours against 500 mls of a 3 Msolution in NaCl.

Dialysis is then carried out against 2-mol portions of 1.10⁻⁴ M HClduring four days, the solution being daily renewed.

By freeze-drying the residue of the dialysis, there are obtained 34milligrams of the polymeric derivative of NAD, λ_(max) 276.5.

The determination of the total NAD bound to the polymer is carried outby the measure of the absorbance at 276.5 nm, assuming an extinctioncoefficient equal to that of the derivative II of NAD (ε18,800 M⁻¹cm⁻¹). The determination of the coenzymically active NAD bound to thepolymer is carried out by quantitative enzymic reduction with alcoholdehydrogenase from yeast in a 0.1 M tris buffer at pH 9 in the presenceof 0.2 M ethanol and 0.005 M semicarbazide hydrochloride.

From the spectrophotometric measure at 340 nm of the as-formed NADHderivative, it appears that 157 micromols of enzymically reducible NADare bound to each gram of polymer, corresponding to 90% of the total NADbound to the polymer.

The thus obtained macromolecularized NAD is coenzymically active withseveral dehydrogenases.

EXAMPLE 6

Preparation of thenicotinoylamide-8-(aminohexyl-sepharose-2-carbonylethylthio)adeninedinucleotide. ##STR8##

500 milligrams of aminohexyl sepharose 4B are allowed to swell with a0.5 M NaCl solution, then washed with 200 mls of 0.5 M NaCl and thenwith water. To the as-obtained gel, slurried in 2 mls of water, thereare added 40 milligrams of nicotinoylamide-8-(carboxyethylthio)adeninedinucleotide. ##STR9## dissolved in 0.5 ml of water and the pH isadjusted to 4.7 with 1 M NaOH. To the slurry stirred at room temperaturewith a mechanical stirrer, there are added 40 milligrams ofN-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride dissolvedin 0.2 ml water and stirring is continued during 24 hours whilemaintaining the pH at 4.7 during the first three hours by adding 0.1 MHCl.

Under the same conditions there are added at 24-hour intervals, threeadditional increments of the carbodiimide solution (40 milligrams in 0.2ml of water) for a total reaction time of 96 hours. The gel is thenfiltered and washed with a 1 M solution in NaCl and 1.10⁻⁴ M in HCluntil the disappearance of the UV absorption (not bound NAD) isexperienced. There are thus obtained 1.8 ml of the polymeric derivativeof NAD in the form of a moist gel, λ_(max) 276.5 nm.

The UV spectrum has been obtained by suspending the gel in an aqueous50% solution of sucrose (weight/weight) which delays the settling of thegel. The determination of the total NAD bound to the polymer is carriedout by the measurement of the absorbance at 276.5 nm, assuming anextinction coefficient equal to that of the derivative II of NAD(ε18,800 M⁻¹ cm⁻¹). The determination of the coenzymically active NADbound to the polymer is carried out by quantitative enzymic reductionwith alcohol dehydrogenase from yeast by suspending the gel in a 50%aqueous solution of sucrose (weight/weight) containing 0.1 M tris.HClbuffer, 0.2 M ethanol and 0.5 M semicarbazide hydrochloride, adjusted toa pH of 9.

From the photometric readings at 340 nm of the NADH derivative, itappears that 21 micromols of enzymically reducible NAD are bound to eachgram of the dry product, that which corresponds to 80% of the total NADbound to the polymer.

EXAMPLE 7

Preparation of thenicotinoylamide-8-(polyethyleneimine-2-carbonylethylthio) adeninedinucleotide phosphate. ##STR10##

To 0.38 ml of a 33% aqueous solution of polyethyleneimine(weight/volume) adjusted to a pH of 5 with conc. HCl, there are added 20milligrams of nicotinoylamide-8-(carboxyethylthio)adenine dinucleotidephosphate ##STR11## dissolved in 0.5 ml of water and 20 milligrams ofN-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride dissolvedin 0.2 ml of water. The reaction mixture, adjusted to a pH of 4.8 byaddition of 2 M HCl, is stirred at room temperature during 24 hours bymaintaining the pH at 4.8 during the first 3 hours by addition of 0.1 MHCl. Under the same conditions there are added 20 additional milligramsof cabodiimide in 0.2 ml of water and stirring is continued during 24additional hours. The reaction mixture, dilute with water to a volume of10 mls, is then transferred to a centrifugation tube and precipitatedwith 10 mls of 1 M phosphate buffer at a pH of 6. Centrifugation iscarried out during 10 minutes at 39,000 g and the solution is strippedof the polymeric precipitate by decantation. To further purify thepolymer, the latter is dissolved in 2 mls of 2 M solution in NaCl and0.05 M in pH 5.5 acetate buffer and the solution thus obtained issupplemented with 8 mls of water and precipitated again with 10 mls of 1M, pH 6 phosphate buffer and centrifuged during 10 minutes at 39,000 g,the polymer being collected by decantation. Such a purificationprocedure is repeated for at least 4 times. The product, redissolved ina 2 M solution in NaCl and 0.05 M in pH 5.5 acetate buffer, is dializedagainst 1-liter portions of 1.10⁻⁴ M HCl during four days, the solutionbeing renewed daily. By freeze-drying the residue of the dialysis thereare obtained 83 milligrams of the polymeric derivative of the NADP,λ_(max) 276.5 nm. The determination of the total NADP bound to thepolymer is carried out by measuring the absorbance at 276.5 nm, assumingan extinction coefficient equal to that of the derivative II of NAD(ε18,000 M⁻¹ cm⁻¹).

The determination of the coenzymically active NADP bound to the polymeris carried out by quantitative enzymic reduction withglucose-6-phosphate dehydrogenase from yeast in 86.3 nM triethanolaminebuffer, pH 7.6, containing 6.7 mM MgCl₂ and 1.2 mM glucose-6-phosphate.From the spectrophotometric measure at 340 nm of NADP derivative asformed, it appears that 23 micromols of enzymically reducible NADP arebound to each gram of polymer, that which corresponds to 20% of thetotal NADP bound to the polymer.

The thus obtained macromolecularized NADP is coenzymically active withseveral dehydrogenase such as for example 6-phosphogluconatedehydrogenase and L-glutamate dehydrogenase.

What we claim is:
 1. Macromolecularized adenine derivatives having thegeneral formula: ##STR12## wherein the radical ##STR13## is derived froma compound selected from the group consisting of nicotinoylamide-adeninedinucleotide, nicotinoylamide-adenine dinucleotide phosphate, adenosinemonophosphate, cyclic adenosine monophosphate, adenosine triphosphate,adenosine and adenine, and the nitrogen atom bound to the CO group ispart of a compound selected from the group consisting of polylysine,omega-aminoalkyl polyanilamides, polysaccharide esters ofomega-aminoalkyl carbamic acids, polyvinylamine, omega-aminoalkyl estersand omega-aminoalkyl amides of polyglutamic acid, aminoalkyl silanizedglass microspheres, and polyethyleneimine. 2.Nicotinoylamide-8-(polyethyleneimine-3-carbonylethylthio)adeninedinucleotide. 3.Nicotinoylamide-8-(polylysine-2-carbonylethylthio)adenine dinucleotide.4. Nicotinoylamide-8-(aminohexyl-sepharose-2-carbonylethylthio)adeninedinucleotide. 5.Nicotinoylamide-8-(polyethyleneimine-2-carbonylethylthio)adeninedinucleotide phosphate.